HNPP Fluorescent Detection Set Troubleshooting

Troubleshooting

Anti-digoxigenin-AP concentration
Decrease concentration of the anti-digoxigenin-AP conjugate. The conjugate can be diluted down to 1:10.000 in buffer 2.

Immunological detection
Reduce incubation time with diluted anti-digoxigenin-AP down to 20 minutes.

Staining
Reduce incubation time or incubation steps.

II. Crystal-like structuresStorage of slide preparations
When storing the stained slide preparations for several weeks, a crystal-like structure might be formed, coating tissues and chromosomes. Thus storage of stained preparations over long periods is not recommended. For repeated analysis we recommend to carry out a new detection reaction using fresh slide preparations.

Labeling
Check the efficiency of your DIG DNA or RNA labeling by comparison to a labeled control DNA or RNA.

Membrane
The membrane quality influences sensitivity and speed of detection. We recommend nylon membranes, positively charged from Roche Molecular Biochemicals. Other types of nylon membranes [e.g., Biodyne A (Pall), Magnagraph (MSI)] are also suitable. Some membranes may cause strong background formation. When using nitrocellulose membranes a reduction in sensitivity must be accepted.

Hybridization
Increase the concentration of DIG-labeled DNA or RNA probe in the hybridization solution, but only to a concentration where background is still low.Reduce the stringency of the washing steps, such as perform the last stringency wash with 0.5 x SSC, 0.1% SDS instead of 0.1 x SSC, 0.1% SDS at +68 °C.

Detection
Increase the concentration of the anti-digoxigenin-AP conjugate to a 1:5000 dilution (150 mU/ml).

Substrate incubation
Incubation at +37 °C of the sealed membrane in HNPP solution yields stronger signals compared to +15 to +25 °C and should be performed in the dark.

Documentation
Use UV lamps of 302 nm; for optimal results adjust two lamps in a position of highest irradiation intensity closely above the membrane (Polaroid camera situated between the UV lamps.) Commonly used transilluminators are also suitable; note that different wavelengths cause signal reduction (254 nm about factor 10; 366 nm about factor 3). The DNA side of the membrane must always face the UV source. Remove the membrane from the hybridization bag. Some types of plastic can partially block the UV light.The type of film may also influence the sensitivity. We have tested and can recommend the Polaroid film 667 b/w.

II. High background

Labeling
Purify DNA/RNA by phenol/chloroform extraction and/or ethanol precipitation before labeling. Make sure that the probe does not contain cross hybridizing vector sequences.

Membrane
Although the protocol is optimized for the use of the nylon membranes, positively charged, some types which are very highly charged can cause background. Lot-to-lot variations in some membranes may also cause problems. When using the recommended function-tested Roche Molecular Biochemicals membrane, these problems are avoided.

HybridizationNote: It can be necessary to decrease the concentration of the DIG labeled DNA or RNA probe.The critical probe concentration limit is the maximal concentration of probe recommended for hybridization, without the incident of background. It can be unique to a membrane lot and brand, and can be determined by hybridizing a blank piece of membrane with increasing probe concentrations. Homologous DNA may also be spotted on this piece of membrane for a test hybridization. Care should be taken not to permit the membranes to dry throughout the whole procedure.

Detection
Decrease concentration of the anti-digoxigenin-AP conjugate. Increase volumes of the washing and blocking solution and duration of the washing and blocking steps.Spotty background may be caused by precipitates in the anti-digoxigenin-AP conjugate: remove by a quick centrifugation step. (Note: Several centrifugation steps can cause a certain loss of material, which must be compensated for by use of larger amounts).